Studies on the uptake of thyroxine (T4) and triiodothyronine (T3) by isolated rabbit fat cells. Adipose tissue may be considered a target tissue for thyroid hormone, based on the observation that the release of free fatty acids for adipose tissue or free fat cells in vitro and particularly the lipolytic response to catecholamines and glucagon is markedly dependent on the thyroid status of the animal. A diminished lipolytic response to norepinephrine has also been found in adipose tissue of hypothyroid human beings. Another important feature of adipose tissue is that a relatively homogeneous population of fat cells can be prepared readily. Isolated fat cells were prepared from perirenal and epididymal fat tissue of male white New Zealand rabbits. To study uptake, a mixture containing 3 -4 x 10 to the 6th power fat cells and 125I-T3 or 125I-T4 in 2 ml total volume Krebs-Ringer phosphate (KRP)-3% ovalbumin buffer, pH 7.4, was gently rotated at 37 degrees C. Radioactive hormone bound to the cells was determined by collection on a 5.0 micron Millipore filter and counting the air-dried filter. Since it appears likely that it is the free form of the hormone which is available for cellular uptake, very low concentrations of T3 and T4, 1.5 x 10 the minus 11th power M, of the order of free hormone concentrations, were used in the uptake studies. The time course of uptake indicated that T3 and T4 uptake by fat cells was rapid with an apparent steady state reached in 5 min. Consequently, subsequent uptake studies were done at 5 min times. T4 is taken up by fat cells about one-half to one-third the extent of T3, depending on the concentration, in the range 2.5 x 10 to the minus 12th power to 1 x 10 to the minus 9th power M hormone concentrations. A Scatchard plot of the binding data indicated the existence of high affinity, low capacity binding sites with a dissociation constant of about 2 x 10 to the 11th power M for both hormones. The estimated maximal binding capacities for T3 and T4 were 158 and 60 molecules per fat cell, respectively.